WO2018129644A1 - Method, reaction system and kit for improving quality of nucleic acid polymerization sequencing - Google Patents

Abstract

Disclosed are a method, a reaction system and a kit for improving the quality of nucleic acid polymerization sequencing. The method for improving the quality of nucleic acid polymerization sequencing of the present application comprises: adding a second nucleotide into a polymerization sequencing reaction solution, a 3' saccharide hydroxyl group of the second nucleotide being provided with a blocking modification but no fluorescence modification.

Description

Method, reaction system and kit for improving quality of nucleic acid polymerization sequencing Technical field

The present application relates to the field of nucleic acid sequencing, and in particular to a method, a reaction system and a kit for improving the quality of nucleic acid polymerization sequencing.

Background technique

Nucleic acid polymerization sequencing, ie, side-synthesis sequencing (abbreviated SBS), refers to the ability to add fluorescently labeled nucleotides during the SBS process (Metzker et. al., Genome Res 15(12): 1767-1776 (2005)). Helps identify template DNA bases (Prober et. al., Science 238:336-341 (1987)) to give DNA sequence information. In recent years, compared with other sequencing methods, polymerization sequencing has been favored for its high throughput and low price. In this sequencing method, a reversible termination and a base with a fluorescent signal are synthesized by a DNA polymerase; specifically, a nucleotide having a modification at the 3' sugar hydroxyl group is used, thereby blocking the addition of other nucleotides, Fluorescent signals to distinguish between 4 different bases. After removal of the blocking and fluorophores, the naturally free 3' hydroxyl group is restored for addition to the next nucleotide and the fluorescent signal is removed to facilitate synthesis and detection of the next base. In SBS, the synthesis efficiency is very high, and in the case of incomplete synthesis in multiple copies, signal confusion will occur, affecting the read length and accuracy of sequencing. Nucleotides with reversible blocking and fluorophores are required to be added in sequencing while synthesis. Due to the increased modification, the molecules of the nucleotides are larger, the molecular structure is special, and the nucleotides are not modified. In contrast, DNA polymerases are less efficient at identifying and synthesizing such modified dNTPs, limiting the read length and accuracy of sequencing.

Summary of the invention

It is an object of the present application to provide an improved method, reaction system and kit for improving the quality of nucleic acid polymerization sequencing.

In order to achieve the above objectives, the present application adopts the following technical solutions:

The present application discloses a method for improving the quality of nucleic acid polymerization sequencing comprising the addition of a second type of nucleotide to the reaction sequence of the polymerization sequence, the 3' sugar hydroxyl group of the second type of nucleotide having a blocking modification but no fluorescent modification.

It should be noted that the key point of the present application is that dNTPs having only blocking modification, that is, a second type of nucleotide, are added to the reaction solution of the polymerization sequencing. The effect of blocking modified dNTPs on DNA polymerase recognition and synthesis is relatively small. Therefore, the addition of the second type of nucleotides can complement the incomplete reaction, increase the efficiency of the synthesis reaction, and reduce the risk of insufficient reaction; And because of the second category Nucleotides are not fluorescently modified, which reduces signal interference caused by unclear fluorescence refraction or unclean fluorescence of the excised cells, which improves the efficiency of excision and reduces the sequencing error rate; thereby improving the quality of sequencing. It can be understood that the key of the method of the present application is that a second type of nucleotide having only blocking modification is added to the reaction solution of the polymerization sequencing, as for other components of the reaction solution, and specific reaction conditions, reference may be made to Some polymerization sequencing methods.

Preferably, the second type of nucleotide is used in the same amount as the first type of nucleotide in the reaction solution, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.

It should be noted that the first nucleotide in the present application is a nucleotide which is normally added in the polymerization sequencing. In the preferred embodiment of the present application, the second nucleotide and the first nucleotide are added in equal amounts. . It can be understood that in some special designs, the amount of the second type of nucleotide can be adjusted according to requirements, but as long as the second type of nucleotide is added, the synthesis reaction efficiency can be improved to a certain extent. The effect of sequencing error rate.

Another aspect of the present application discloses the use of the method of the present application in nucleic acid sequencing. The method of the present application is applicable to the sequencing of nucleic acid sequences of the human genome and other animal, plant and microbial species; the main applications include sequencing of WES, WGS, RNA, DNA, etc.; in one embodiment of the present application, the method of the present application is applied to the large intestine DNA sequencing of Bacillus.

A further aspect of the present application discloses a nucleic acid sequencing method, which comprises simultaneously adding two types of nucleotides to a reaction solution in a process of sequencing while synthesizing, the first nucleotide being a 3' sugar hydroxyl group and having a blocking effect. Modified and fluorescently modified nucleotides, the second type of nucleotide is a 3' sugar hydroxyl group having only a blocking modification but no fluorescent modification.

It can be understood that the method for improving the quality of nucleic acid polymerization sequencing of the present application can be applied to various sequencing platforms based on edge synthesis sequencing, and the application only needs to be added to the reaction solution without changing the sequencing conditions and parameters. The second type of nucleotide can be.

Another aspect of the present application discloses a reaction system for improving the quality of nucleic acid sequencing, comprising a reaction solution for sequencing while synthesizing, wherein a second type of nucleotide is added to the reaction solution, and a third nucleotide of the second type is added. The saccharide hydroxyl group has a blocking modification but no fluorescent modification.

Preferably, the reaction solution comprises a reaction buffer, a DNA polymerase and a first type of nucleotide, and the first type of nucleotide is a 3' sugar hydroxyl group having both a blocking modification and a fluorescent modification.

A further aspect of the present application discloses a nucleic acid sequencing kit comprising the reaction system of the present application.

It can be understood that the key inventive idea of the present application is to add dNTPs having only blocking modification, that is, a second type of nucleotide, in the reaction solution which is synthesized while synthesizing, so as to improve the synthesis efficiency; The present application further proposes a reaction system for improving the quality of nucleic acid sequencing, that is, a reaction solution prepared by sequencing while adding a second type of nucleotide. Using the reagent system of the present application, Can effectively improve the quality of sequencing. Similarly, for ease of use, the reagent system of the present application can also be made into a nucleic acid sequencing kit.

Due to the adoption of the above technical solutions, the beneficial effects of the present application are:

The method for improving the quality of nucleic acid polymerization sequencing of the present application adds dNTPs having only blocking modification to the reaction liquid, and since such dNTPs have no fluorescent modification, it is more conducive to DNA polymerase recognition and synthesis, and can supplement the incomplete reaction portion. Increasing the efficiency of the synthesis reaction and reducing the risk of insufficient reaction; at the same time, because there is no fluorescence modification, the signal interference caused by the unclear fluorescence removal or the uncleaned fluorescence of the resection is reduced, and the resection efficiency is improved and the reduction is improved. Sequencing error rate.

DRAWINGS

1 is a schematic structural view of a second type of nucleotide in the embodiment of the present application;

2 is a schematic structural view of a first type of nucleotide in the embodiment of the present application;

3 is a fluorescence signal change curve of each cycle in the embodiment of the present application;

4 is a sampling quality change curve of each cycle in the embodiment of the present application;

Figure 5 is a graph showing the variation in sequencing error rate for each cycle in the examples of the present application.

detailed description

The key to sequencing while synthesizing is to add dNTPs with both blocking and fluorescent modifications in the reaction solution. However, the inventors of the present application have conducted extensive experiments and studies to find that blocking and fluorescently modified dNTPs may affect The recognition and synthesis efficiency of DNA polymerase limits the read length and accuracy of polymerization sequencing. After intensive research, it is found that fluorescent modification is the main factor affecting the recognition and synthesis efficiency of DNA polymerase; therefore, the present application proposes to add dNTPs having only blocking modification on the basis of the existing polymerization sequencing reaction solution; On the one hand, the second type of nucleotide can be used to supplement the incomplete part, which guarantees the synthesis efficiency and reduces the risk of insufficient reaction; on the other hand, since the second type of nucleotide has no fluorescence modification, the fluorescence excision is also reduced. Unclean, or signal interference caused by unclear fluorescent elution, improves the efficiency of resection and reduces the sequencing error rate.

The present application will be further described in detail below by way of specific embodiments and the accompanying drawings. The following examples are only intended to further illustrate the present application and are not to be construed as limiting the invention.

Example

The nucleic acid sequencing method of this example was performed on a BGISEQ-500 sequencing platform, and the sequenced sample was an E. coli standard library. The specific plan is as follows:

(1) using the sequencing platform of BGISEQ-500, and using the sequencing reagents matched with the platform according to the instructions;

(2) According to the formulation of Table 1, the dNTP 1 mixture is prepared, that is, the first nucleotide; the first nucleotide of this example, as shown in FIG. 2, has both a fluorescent dye and a blocking group on the nucleotide. Wherein A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.

Table 1 dNTP 1 mixed solution formula

dNTP 1 mixture Final concentration (nmol/L) dCTP_1 100 dGTP_1 100 dATP_1 100 dTTP_1 100

In the table, dCTP_1 refers to cytosine nucleotides with both blocking and fluorescent modifications, dGTP_1 refers to guanine nucleotides with both blocking and fluorescent modifications, and dATP_1 refers to both blocking and fluorescent modifications. The adenine nucleotide, dTTP_1 refers to a thymidine nucleotide having both blocking and fluorescent modifications.

(3) According to the formulation of Table 2, the dNTP 2 mixture is configured, that is, the second type of nucleotide; the second nucleotide of this example, as shown in Figure 1, has only a blocking group on the nucleotide, and no fluorescence. A dye, wherein A, T, G, and C represent adenine nucleotides, thymidine nucleotides, guanine nucleotides, and cytosine nucleotides, respectively.

Table 2 dNTP 2 mixed solution formula

dNTP 2 mixture Final concentration (nmol/L) dCTP_2 100 dGTP_2 100 dATP_2 100 dTTP_2 100

In the table, dCTP_2 refers to a cytosine nucleotide having only a blocking modification, dGTP_2 refers to a guanine nucleotide having only a blocking modification, dATP_2 refers to an adenine nucleotide having only a blocking modification, and dTTP_2 is Refers to a thymidine nucleotide that only has a blocking modification.

(4) Using the BGISEQ-500 platform, compare the addition of dNTP 2 mixture to achieve supplementary reaction. For the effect, and keep the other reagents unchanged, take out the reagents in the #5 and #6 holes of the kit and replace them with the reagents needed for the experimental group;

(5) Prepare the synthetic reagent 1 according to Table 3, mix well and set aside;

Table 3 Synthetic Reagent 1 Formulation Table

Synthetic reagent 1 Dosage (mL) Reaction buffer 48 DNA polymerase 1 dNTP 1 mixture 1

(6) Prepare the synthesis reagent 2 according to Table 4, mix well and set aside;

Table 4 Synthetic Reagent 2 Formulation Table

Synthetic reagent 2 Dosage (mL) Reaction buffer 48 DNA polymerase 1 dNTP 2 mixture 1

(7) Add the same amount of synthetic reagent 1 in the #5 hole position and #6 hole position of the control kit, and add the synthetic reagent 1 and the #6 hole to the synthetic reagent 2 in the kit #5 hole of the experimental group. The ratio of the two reagents is 1:1.

(8) SE50 sequencing was performed using the BGISEQ-500 platform, the experimental group with the synthetic reagent 1 was added as the control group, and the experimental group with the synthetic reagent 2 was added as the experimental group, and the fluorescence signal curves of each cycle in the two groups were statistically analyzed. Sequencing quality change curve for each cycle and sequencing error rate curve for each cycle.

The sequencing results are shown in Figures 3 to 5. Among them, the fluorescence signal change curve of each cycle is shown in Fig. 3. In the figure, the abscissa is the sequencing read length, the ordinate is the sequencing signal, “○” is the curve of the experimental group, and “▲” is the curve of the control group; The results showed that with the increase of sequencing cycle, the sequencing signal decreased slowly, and the fluorescence signal of the experimental group decreased more slowly than the control group, indicating that sequencing synthesis and excision were more complete.

The sequencing quality variation curve of each cycle is shown in Fig. 4. In the figure, the abscissa is the sequencing read length, the ordinate is the sequencing quality value, “○” is the curve of the experimental group, and “▲” is the curve of the control group; It is shown that with the increase of the sequencing cycle, the quality of sequencing is reduced, and the sequencing quality of the experimental group is slower than that of the control group, indicating that the sequencing quality of the experimental group is higher.

The sequencing error rate curve for each cycle is shown in Figure 5. In the figure, the abscissa is the sequencing read length. The ordinate is the sequencing error rate, “○” is the curve of the experimental group, and “▲” is the curve of the control group; the results show that with the increase of the sequencing cycle, the sequencing error rate begins to increase, and the error rate of the experimental group increases faster than the comparison. The group should be slow, indicating that the experimental group has a lower error rate.

It can be seen that the addition of the dNTPs having the blocking modification, that is, the second type of nucleotides of the present application, in the polymerization sequencing reaction solution can effectively improve the sequencing quality and reduce the sequencing error rate.

The above content is a further detailed description of the present application in conjunction with the specific embodiments, and the specific implementation of the present application is not limited to the description. It will be apparent to those skilled in the art that the present invention can be made in the form of the present invention without departing from the scope of the present invention.

Claims (9)

A method for improving the quality of nucleic acid polymerization sequencing, comprising: adding a second type of nucleotide to a reaction solution for polymerization sequencing, wherein the 3' sugar hydroxyl group of the second nucleotide has a blocking modification but no fluorescence Modification. The method according to claim 1, wherein said second nucleotide is used in the same amount as the first nucleotide in the reaction solution, and said first nucleotide is a 3' sugar. The hydroxyl group has both a nucleotide that blocks modification and fluorescence modification. Use of the method according to claim 1 or 2 in nucleic acid sequencing. The nucleic acid according to claim 3 comprises DNA or RNA. Use of the method according to claim 1 or 2 in human genome, animal, plant and/or microbial nucleic acid sequencing. A nucleic acid sequencing method, which comprises the steps of simultaneously adding two types of nucleotides in a reaction mixture during the sequencing process, wherein the first nucleotide is a 3' sugar hydroxyl group and has blocking modification and fluorescence. A modified nucleotide, the second type of nucleotide is a nucleotide having a 3' sugar hydroxyl group having only a blocking modification but no fluorescent modification. A reaction system for improving the quality of nucleic acid sequencing, comprising a reaction solution for sequencing while synthesizing, characterized in that: a second type of nucleotide is added to the reaction solution, and 3' of the second type of nucleotide The saccharide hydroxyl group has a blocking modification but no fluorescent modification. The reaction system according to claim 7, wherein the reaction solution comprises a reaction buffer, a DNA polymerase and a first type of nucleotide, and the first type of nucleotide is a 3' sugar hydroxyl group and has a hindrance. Broken and fluorescently modified nucleotides. A nucleic acid sequencing kit comprising the reaction system of claim 7 or 8.

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